Measurement, analysis and isolation of desirable cells is a well established goal of much of industrial microbiology and biotechnology, with many important applications in fields such as clinical microbiology, cancer diagnosis and treatment, environmental science, food safety, toxicology, and research and development in basic and applied biology.
The importance of such isolation and prior methods for accomplishing isolation are well known (see, for example, Queener and Lively in Manual of Industrial Microbiology and Biotechnology, Demain and Solomon (Eds.), American Soc. Microbiol., Washington, D.C., 155-169, 1986; White et al, ibid, pp. 24-31). Selection strategies, which are based on preferential growth and recovery of desired cells, are often possible. For example, selection can be accomplished by genetically manipulating cells such that a desired trait is coupled with resistance to antibiotics or to the ability to grow in the absence of certain compounds. However, there are many cases in which selection does not work well or does not work at all. In such instances, cells are often isolated by screening, i.e. by methods wherein cells are cloned, identified and one or more desirable properties measured, such that a particular clone is identified, physically removed, and thereby isolated.
One well established application of screening methods for microorganisms involves cells which have been exposed to mutagenic conditions, or to genetic splicing procedures, such that a large population of cells results, but of which only a very small number of cells are successes, e.g. cells which both grow and produce a desired molecule at a high level. Plating of such cells on petri dishes such that separated colonies arise is then followed by chemical assays. In such assays, a successful colony which is scored positive (+) by use of radioimmunoassay, or is colored through use of a colorimetric indicator. The number of cells in such colonies is typically large. For example, in the case of cells which are microorganisms such as bacteria, such colonies typically contain between 10.sup.7 and 10.sup.9 cells. Cells from successful colonies are then removed and suspended in a medium such that the number of cells can be enumerated, and the accumulated amount or the production rate of a desired molecule can be quantitatively determined. As is evident from this brief description, this type of procedure requires several steps. Currently, the procedure is generally carried out using relatively macroscopic entities and manual procedures, such as petri dishes (for initial colony formation), visual inspection (for identifying positive colonies), physical manipulation (for colony removal and suspension), physical measurement (for cell enumeration by light scattering or other means) chemical assay (for determination of molecule concentrations at different times) and calculation (for comparison of molecule production rate to known rates).
Screening is often much more difficult when a producing strain already exists, and the task is to seek rare mutants which produce the same molecule, but at higher rates, or to higher final concentrations. In such cases, the first step of scoring positive cells is unnecessary, but then all cells must be quantitatively assayed for relative productivity. This is generally a very time consuming task. Because of the several macroscopic manipulations and assays, a typical screening task can involve formation of many initial colonies and significant manual labor.
The importance of measurement of biological entities, and the prior methods for accomplishing such measurement are also well known, with applications in fields such as clinical microbiology, cancer diagnosis and treatment, environmental science, food safety, toxicology, and research and development in basic and applied biology. Measurements relating to biological entities such as cells, virus, nucleic acids and antibody-antigen complexes are of general interest. Although a significant number of assays and tests are directed towards virus, nucleic acids and antibody-antigen complexes, the most well established methods for determining measurements of such biological entities relates to measurements of cells. For this reason, the main features of prior methods for the process for manipulation and measurement of non-aqueous surrounded microdroplets are presented with an emphasis on prior methods for determining cell growth.
Present cell analysis methods involve two major classes of assays. The first class rapidly detects and identifies specific cells directly from a primary sample, but does not determine cell viability. The most widely used in this class are specific ligand binding assays, e.g. immunoassays and genetic probes. However, they require many cells, and do not distinguish between dead and viable cells. This restricts their use to samples in which sufficient numbers of cells are present, and to determinations in which direct assessment of the physiological state of the cell is irrelevant. The second class of assays is used for viable cell determinations either directly using the primary sample, or using a subculture of the primary sample. The most traditional and widely used method is the plate count, which allows determination of single cell viability, based on growth, under many test conditions (see, for example, Hattori The Viable Count: Quantitative and Environmental Aspects, Brock/Springer, Madison, 1988). An important attribute of viable plate enumeration is that time required to obtain a determination is independent of the concentration of the cell in the sample, because formation of each colony proceeds from an initial single cell. The major disadvantage is its slowness, as typical determinations require one-half to several days, and are also labor- and materials-intensive.
The disadvantages of viable plating can better be appreciated by drawing attention to its basic attributes. Viable plating is a well established, important method for qualitatively determining the growth of cells, particularly the presence or absence of growth for given conditions, and is often based on the growth of initial cells into distinct colonies. Viable plating typically involves the spreading of a suspension of cells onto the surface of a gel-containing petri dish, with or without the pouring of a gel layer over the first gel surface. The gels are provided with nutrients, such that following an incubation period at a suitable temperature, many generations of growth occur. This leads to formation of visible colonies. For many microorganisms formation of visible colonies requires growth for 22 to 30 generations and therefore produces colonies containing 10.sup.7 to 10.sup.9 cells. (See Sharpe, in Mechanizing Microbiology, A. N. Sharpe and D. S. Clark (Eds.) Charles C. Thomas, Springfield, 19-40, 1978). Although conventional viable plating leads to formation of colonies, and thereby provides a basis for counting viable cells by counting colonies, the presence or absence of colonies only allows inference that the conditions present in the gel support do or do not support growth. For this reason, conventional viable plating is not well suited to quantitative determinations such as cell growth rate and lag time, because viable plating based on visual inspection counts the number of colonies formed, but does not determine how the cellular material or amount of cellular constituents in the colonies varies with time. An additional complication arises because the nutrient and metabolite concentrations within a colony comprise a microenvironment, which generally changes with time in an unknown way as microcolonies increase to form larger colonies with many cells in close proximity. The microenvironment within a large colony can also have significant heterogeneity of chemical composition within the microcolony, so that different cells within a large colony experience different growth conditions. Further, although some methods are based on a straightforward extension and application of scanning optical methods for determination of optical properties of colonies on or in gel slabs, such methods suffer from relatively large cost and size, and, because of the relatively large gel slab size, do not allow incubation conditions to be changed rapidly at the site of the cells within the gel. (See Glaser in New Approaches to the Identification of Microorganisms Proceedings of a Symposium on Rapid Methods and Automation in Microbiology, C.-G. Heden and T. Illeni (Eds.), Wiley, New York, 3-12, 1975).
Instrumented methods for rapidly determining cell or culture growth and/or metabolic activity have been developed which only partially address the limitations of the viable plate assay. These include optical techniques for growth determination such as those which measure the change in light scattering due to many cells in a liquid suspended culture (See, for example, Edberg and Berger, in Rapid Methods and Automation in Microbiology and Immunology, K. O. Habermehl, Ed., SpringerVerlag, Berlin, 215-221, 1985), and a variety of metabolic activity based techniques which measure changes due to many cells in an analyzed sample. Examples of these include changes in extracellular pH (See, for example, Cowan and Steel's Manual for the Identification of Medical Bacteria, Cambridge University Press, Cambridge, 1974; Manual of Methods for General Bacteriology, P. Gerhardt, (Ed), American Society for Microbiology, Washington, 1981 ), carbon dioxide release (see, for example, Courcol et al., J. Clin. Microbiol., 25: 26-29, 1986; Manca et al., J. Clin. Microbiol., 23: 401-403, 1986), electrical impedance (see, for example, Stewart, J. Exp. Med., 4: 235-245, 1899; Eden and Eden Impedance Microbiology, Research Studies Press, Letchworth, 1984; Hadley and Yajko, in Instrumental Methods for Rapid Microbiological Analysis, Nelson (Ed.), VCH, Weinheim, 193-209, 1985; Bishop and White, J. Food Protect, 49: 739-753, 1986), chemiluminescence (see, for example, Neufeld et al., in Instrumental Methods for Rapid Microbiological Analysis, Nelson (Ed.), VCH, Weinheim, 51-65, 1985; Rapid Methods and Automation in Microbiology and Immunology, Habermehl (Ed.), Springer-Verlag, Berlin, 1985) or fluorescence (see, for example, Rossi and Warner in Instrumental Methods and Automation in Microbiology and Immunology, Habermehl (Ed.), Springer-Verlag, Berlin, 1985). A disadvantage of all such metabolic activity methods is that they are based on combined effects of a large number of cells. Therefore, this generally requires an initial process, based on plating, to obtain initial colonies for purposes of inoculation of the analyzed sample, such that the determinations based on many cells at least are based on a monopopulation, i.e. a population comprised norminally of the same type of cells. For this reason, although a total population cell determination may itself be rapid, it is generally preceeded by a viable plating method, or its equivalent, which is slow. Thus, the total analysis time, counted from receipt of a primary or non-plated sample to a cell growth determination, is the sum of both, and therefore still long.
Further, because such determinations are based on the combined effect of a large, but unknown, number of cells, such total population determinations do not actually yield a count. In contrast, determinations based on many individual measurements, each associated with an initial single cell, can yield a count.
Finally, because these total population methods are based on the combined effects of many cells, the time required for a determination becomes significantly longer as the number of cells decreases, i.e. as the sample's cell concentration decreases.
Similarly, prior use of flow cytometry for cell growth measurements (see, for example, Hadley et al. in Instrumental Methods for Rapid Microbiological Analysis, Nelson (Ed.), VCH, Weinheim, 67-89, 1985) is limited, because conventional use of flow cytometry performs measurements on individual cells, or clumps of cells which naturally adhere, in an aqueous liquid suspension, and therefore does not have the capability to measure colony formation. For this reason, prior use of flow cytometry can only measure total numbers of cells in a volume in order to determine average growth, must also, therefore, involve a careful volume measurement, and is dependent on the signal-to-noise ratio of single cell measurements. This signal-to-noise ratio is less than satisfactory for many measurements (see, for example, Shapiro Practical Flow Cytometry, R. Liss, New York, 1985; Hadley et al. in Instrumental Methods for Rapid Microbiological Analysis, Nelson (Ed.), VCH, Weinheim, 67-89, 1985).
Likewise, quantitative microscopy and image analysis combined with conventional gel preparations, such as gel slabs, petri dishes and the like, although capable of determining colony formation, is tedious in manual versions, and conventional gel slabs, petri dishes and the like cannot provide physical manipulability or a sufficiently fast characteristic diffusion time within the gel. Thus, cells cannot be rapidly and conveniently exposed to different growth conditions, such as rapid changes in concentrations of nutrients, drugs, hormones, enzymes, antibodies and other chemicals. In addition, conventional gel slabs, petri dishes and the like cannot be readily manipulated physically because of their size, and therefore cannot be readily used for exposure of gel-entrapped cells to in vivo conditions.
Previous studies of gel microdroplets have demonstrated that the suspension of gel microdroplets (GMDs) in a non-aqueous fluid such as mineral oil results in retention of microbial metabolites such as metabolic acids. This in turn allows detection and enumeration of acid-producing microorganisms through the use of colorimetric and fluorescent pH indicators (Weaver et al., Ann. N.Y. Acad. Sci., 434: 363-372, 1984; Williams et al, Ann. N.Y. Acad. Sci. 501: 350-353, 1987). In the previous work, observation of pH changes by optical means such as colorimetry or fluorescence was accomplished using fluorescence microscopy. However, such prior use demonstrated that insignificant chemical communication of highly water soluble compounds, such as metabolic acids, occurs between GMDs even when they are almost contacting. Thus, such GMDs are found to be essentially chemically isolated, and cannot readily have the chemical composition of the aqueous interior of such GMDs altered or modified.
An attribute of these previous GMD methods is that GMDs surrounded by mineral oil (non-aqueous surrounded GMDs) were provide a chemical isolation of GMDs, such that many chemicals are retained within GMDs. This retention within the very small volume of a GMD is the basis for an important class of measurements on individual cells, individual enzyme molecules, microcolonies, and the like. In this previous use of GMDs, chemical retention is achieved by surrounding GMDs by a non-aqueous fluid which has solubility properties which essentially exclude the dissolution of many chemicals, particularly charged or ionic species such as acids. For this reason, such chemicals do not significantly partition into the non-aqueous fluid, and cannot therefore be transported through the non-aqueous fluid by a combination of diffusion and convection.
Although this is highly desirable in order to retain the extracellular products of biologically active entities within a GMD, and also to provide or retain the chemical assay reactants used within a GMD as the basis of an extracellular and/or intracellular assay, the previous use has resulted in an inability to alter the chemical composition, for chemicals insoluble in the non-aqueous fluid, of the GMD interior once the GMDs were created and surrounded by a non-aqueous fluid.
It would, however, be highly desirable to be able to alter the chemical composition of GMD interior fluids at one or more times after the GMDs have been surrounded by a non-aqueous fluid, under controlled conditions, and without the necessity of removing the GMDs from the non-aqueous fluid followed by a re-suspension or re-surrounding of the GMDs by a non-aqueous fluid.